Project Abstract Lipid-bounded organelles are touted as a defining feature of eukaryotes and one which is absent from the architecturally primitive cells of bacteria. However, numerous bacteria use lipid-bounded organelles to execute essential, and at times toxic, biochemical reactions in a compartmentalized fashion. My group uses the magnetosome organelles of magnetotactic bacteria as a model for understanding the mechanistic basis of organelle formation and function in bacteria. Magnetosomes are lipid-bilayer invaginations of the cell membrane with a unique protein content, within which nanometer-sized iron-based magnetic crystals are produced. Individual magnetosomes are arranged into a chain with the help of an actin-like cytoskeleton, thus allowing magnetotactic bacteria to use geomagnetic fields as a simple guide for low oxygen environments. The cell biological features of magnetosomes make them ideal for understanding the molecular basis of organelle biogenesis in bacteria and, perhaps, uncover the evolutionary origins of eukaryotic organelles. The magnetic and physical properties of magnetosomes make them attractive targets for the development of biomedical applications including their use as contrast agents for magnetic resonance imaging, as drug delivery vehicles and as a medium for hyperthermic killing of tumor cells. In addition to magnetosomes, my group has recently discovered a novel iron-accumulating lipid-bounded organelle named the ferrosome. Ferrosomes are formed through the action of a small number of genes and are found in diverse bacteria including resident members of the gut microbiome and opportunistic pathogens. The research program outlined in this proposal will leverage the expertise and existing knowledge within my group to explore the most critical areas of magnetosome and ferrosome biology. First, we will focus on the cell biological mechanisms that allow for formation and subcellular organization of magnetosomes. We will define the minimal components required for the biogenesis of the magnetosome membrane, uncover the modes and dynamics of protein localization to magnetosomes and study the biochemical and biophysical characteristics of the actin-like cytoskeleton required for organization of magnetosomes. Second, we will investigate the mechanisms of iron biomineralization within magnetosomes. We will uncover the interactions, activity and function of proteins implicated in the nucleation and growth of magnetic particles and will develop simplified in vitro systems to define the kinetics and chemical requirements for biomineralization. Finally, we will use this project period to develop ferrosomes into an alternate and robust model for the study of bacterial organelles. We will determine the mechanisms of ferrosome membrane biogenesis, protein sorting and iron transport and in parallel define their physiological function in relevant microorganisms. The combination of these approaches will shed light on the evolution and mechanistic diversity of bacterial organelles while providing a more rational basis for their use in applied settings.